12.6.12 Worked with ling to assay suman's protein.
The measured absorbance for 410nm was 0. Since other people had similar results it could be reasoned that the cause for this would be: 1.multiple times of defrosting 2.the glycerol mix was bad 3. it was not stored in glycerol at all
Afterwards we measured Suman's protein STSP 2 more times. the first time it did not work because the values for the uM and molecular weight were incorrect, so the addition of tris base was incorrect. The second time, the correct values were entered, and that is when the above conclusions were made.
12-5-12
enzyme assay.JPG
Enzyme assay with FtHap; Negative results as there is no absorbency measured at 410 nm which is the wavelength where the protein should present itself if there was indeed protein activity when introduced with the pNPP. reasons could be :
1. improper technique with pipetting and use of the wrong cuvette
2. the protein was inactive due to denaturing
3. not enough of the protein within the solution. The values (absorbance) were too small to consider using for inhibition. During the collection of absorbance, the samples got mixed up. Also there isn't a linear function as the concentrations of enzyme increased. Also, one of the samples was thrown out because it was skewed from the rest of the data.
12-4-12
Conducted enzyme assays with ling and rishi on the surrogate FtHap. Still getting inconclusive results with the redtide machine. More assays will be run in order to verify whether or not the issue is technique or the protein.
Conducted virtual screening 2nd run - results:
Virtual screening 2nd run results
The results shown here are the top 10 ligands selected by the GOLD program out of 400 compounds from the HF9PlatesPlates library. Compounds HTS09290, SEW02685, and HTS08822 are the top 3 ligands from the selection of 400 and would make the best potential drug candidates out of the compounds screened. The compound 1 shows much promise with a fitness score of 99.23%.
Week 14:
11.30.12
Started work on virtual screening.
11.29.12
Worked on assaying / measuring concentration samples
Worked on swiss model:
11.27.12
Worked on purification protocols - used GE purification tubes in combination with the beckman centrifuge in order to remove water and extract a pure sample. Sample found to be roughly around 0.98 mg/ml.
week 13:
112612 - ?? . Dr B
Characterized the surrogate with rishi - Nanodrop/concentration pulled from rishi's vds page.
protein char.png
Lane 1: Skipped Lane 2: 10-170kDa Protein Ladder Lane 3: Sample 1, Cell lysate after induction Lane 4: Sample 2, Soluble fraction Lane 5: Sample 3, Flow through Lane 6: Sample 4, Wash Lane 7: Elution 1 from Trial 1 (Tube A) Lane 8: Elution 2 from Trial 1 (Tube B) Lane 9-10: Skipped Some of Elution 2 spilled into Lane 9 causing faded bands. Also, Cell Lysate before induction was not inserted into Lane because not available.
Elution 1 (Concentration of 1.40 mg/mL): Tube A from Trial 1
Elution 1ubeB_RDvds.jpg
Elution 1ubeB_RDvds.jpg
Elution 2 (Concentration of 0.11 mg/mL): Tube B from Trial 1
Elution 1tube A_RDvds.jpg
Elution 1tube A_RDvds.jpg
Elution 1 (Concentration of 1.08 mg/mL): Tube C from Trial 2
Elution 2tubeC_RDvds.jpg
Elution 2tubeC_RDvds.jpg
Elution 2 (Concentration of 0.10 mg/mL): Tube D from Trial 2
Elution 2tubeD_RDvds.jpg
Elution 2tubeD_RDvds.jpg
Week 12:
11.18.12
Assisted Rishi on Sonification steps.
When entered onto blast, there was not enough of a match up to give conclusive results.
Week11:
Paul - make sure you use the right primers for you sequencing. Not sure why you are getting unreadables. Maybe show me you output form one of them - post it here on Wiki. -- Dr. B 11/19/12
11.15.12
Morning to mid day was spent with the continued growth of the Fthap colonies in 2L flask ; 4 out of the 6 flasked were used due to space constraints. Every hr I measured OD for the flasked until 0.5 absorbency at 600 nm was reached. Afterwards I added ITPG and left the flask in the shaker for 4 hours to allow it to grow. Subsequent centrifugal was done by ling and rishi after the 4 hour period.
11.14.12
I stayed over night with ling to work on growing up the fthap colonies for surrogate protein expression. 6 flasks in total were left in the shaker over night.
11.12.12
I looked over the data recieved from the core and checked against blast was inconclusive. There was a large number of N and unreadable nucleotides in the sequence. I will grow more bacteria this week and resend samples for sequecing to verify my results.
As well I will now start on the surrogate protein expression.
Week10:
11.9.12
I am currently awaiting results of DNA sequencing.
11.8.12
Nanodrop concentration results : Samples 4 and 9 show really weak absorption/concentration. The reasons for this could be that those colonies did not grow too well in the media, improper growing technique, or improper minipreping.
Next step would be to prepare the samples for sequencing.
11.7.12
Worked on miniprep of the colonies and extraction of dna.
11.6.12
I prepared 9 colonies for master plate production and grew up 9 tubes.
My everything plate has produced colonies - there are roughly around 60 colonies. My next step is to move onto picking a couple of colonies and proceed to making a master plate and producing some bacteria for sequencing.
My every thing plate - roughly 61 colonies
Week9:
11.2.12
I have decided to employ a hail mary approach with my own samples while working with ling and rishi to help them get their samples to cloning. What I have done is mixed together 3-4 months worth of my own personal leftover and unused lmstpp and bsa-4 samples that were properly labeled into one tube of accepting plasmid and one tube of the insert. This produced a high concentration of plasmid to insert. I also ran a gel check to see if the samples were still use able. On a 100b ladder, both the insert and the accepting vector seem to be within the right size, length, and cut amounts. When plating I made a 20:20 ratio of accepting to insert - and added 50 ul of the bacteria to the mixture while prepping. As well checking the nanodrop readings, while the absorbances are high, the wavelengths they each respectively peak at are correct when compared to past samples.
This are the lmstpp samples from the last 4 months mixed together into 1 tube.
This is the bsa-4 accepting vector from the last 4 months mixted together into one tube.
Shared a gel with stephanie - the ladder is 100b at the very end. Lane 3 is the accepting vector - it is faint but you can distinctly see 2 cuts. Lane 4 is the insert which is just underneath the 1000 bp amount which makes it the correct length. The last sample before the ladder is rishi's prepared plasmid
11.1.12
Worked on PCR^2 and clean up with ling.
middle lane is 100b ladder. To the left and right are the clean up samples
10.31.12
Made primer dilutions for the master stock and for working solutions. Attempted to do a midiprep clean up - failed horribly due to incorrect tubes being used.
10.29.12
Prepared samples for additional people for leishmania target.
week8:
10.25.12
Checked on the plates created - no colony growth present. I will attempt to start back at the plasmid and insert preparation step.
10.23.12
By the end of this day I was able to clean up the pcr samples, prepare the plasmid, and transform and plate the bacteria.
PPVDS gel insert 40ng/ul concentration for sample that was through pcr clean up
Regular sample of target after clean up - I made a concentrated solution during clean up by combining 8 tubes extract into 50 ul elution.
Plasmid sample 1 after clean up
plasmid sample 2 after clean up - possibly impure due to weird readings at the 240 nm wavelength
sample 3 plasmid after clean up - 19.6 ng/ul
plasmid sample 4 after clean up - concentration 15.7 ng/ul
10.20.12
Sample prepared from gel extraction- concentration is 41.8 ng/ul - I will preform pcr clean up later.
Sample for gel clean up :lane 1 is 1 kb ladder, lane 2-9 50 ul of pcr^2 sample to be extracted
regular gel check for 8 samples for regular pcr clean up procedure, lane 1 is 1kb ladder, lanes 2-10 are each lmstpp genes.
Week 7:
102112- Paul, ok - show some data/images. -- Dr. B
10.18.12
Worked on PCR^2 ; I created 16 samples, half for gel extraction and the other half for regular pcr clean up procedure.
10.16.12
continued work on the virtual refresher assignment.
10.15.12
Worked on acquainted self with virtual screening program. Looked over and discussed results of the DNA sequencing and compared it with the online nucleotide BLAST program. Out of the 14 samples sent only 3 had any sort of match up with the gene of interest. Of the three none of the samples had the right size to continue onto the next phase - it can be assumed that only a fragment of the insert actually got into the plasmid. Due to this I will have to restart back at the secondary PCR procedure and try to adjust the protocol to see if theres any change in results.
Week 6:
101612 -Paul - ok good job. Hopefully your cloning works out! - Dr. B
10.13.12
Currently working on virtual screening protocols.
10.12.12
The cleaned up plasmids had concentrations determined through the use of the nanodrop machine. The ranges are from 10-22 ng/ul for plasmid concentration. Concentration seems medium to low . 14 samples were sent to sequencing. 12 were had using the forward primers, and the 2 samples with the highest concentration were used with the reverse primers. Still awaiting results.
10.9.12
Colonies were grown up in snap tubes and grown up over night. Procedures for the day included spinning down and using the miniprep clean up kit to extract the plasmid.
10.8.12
Plates grew up successfully over the weekend , a nice amount of colonies are present on each of my plates with out too much of a lawn. Colony count seems to be in the 10's. I will proceed to master plating and growing up in tubes.
Plate A1 has a ratio of 2:1 for plasmid to bacteria. Individual colony count around 50-60.
Plate B1 - had a nice amount of individual colonies - I accidently flipped the plate leading to the condensation moving around the plate. For growing purposes I only selected bacteria from individual colonies.
Plate A2 has 0 colonies. Possible incorrect use of collirollers.
Colony count around 20 ; Plate B has a 1:1 ratio of bacteria and the plasmid.
Created master plate with 12 colonies selected ; 4 colonies from the 3 plates produced. the first 4 from plate A1, the second 4 from B1, and the last 4 from B2
Week 5:
100912 - Paul - I think that your PCR^2 bands need to be a lot bigger in order to have enough to go on to cloning. Does the gel of PCR^2 correlate to the amounts that you get in Nanodrop. Because the nanodrop vaules seem good for the PCR^2 but the bands are weak. Also, you should have multiple wells of PCR^2 - not just one or two , right? - DR. B
Also, try to crop your gels better to get rid of the black space.
10.3.12 ( BSA4 cutting and prepartation - I made 2 preparations of the solution so I could make 2x the plates)
Nanodrop of bsa-1 concentration 21.9 ng/ul
nanogram- concentration at 18.8 ng/ul
Gel of cut bsa4- cut with bsaI - despite the low contrast there is definately a cut as there are 2 bands each.
10.2.12 ( I redid my PCR^2 results on gel)
lane 1 is 1kb ladder, lane 2 and 3 have the tail end primers and the oligoprimers. Despite the resolution and contrast there is definate amplification in the right position. I will continue into transformation.
Spectrometer of oligoprimer concentration : concentration found to be at 65.8 ng/ul
Nanodrop for Tail end primers - concentration at 98.5 ng/ul
Week 4:100112 - Paul, yeah - make sure there is something there before proceeding. -- Dr. B
9.25.12
This is the concentration for the PCR^2 product after clean up with sigma kit. Concentration is 30.6 ng/ul with 260/280 at 1.81 and 260/230 at 1.40. I will wait until the second gel has run before continuing into transformation.
Ran a gel on PCR^2 ; no detection on lane 2. I will rerun another gel.
Week3:
paul - Great. And the 'tail primers' on the left seem to make a larger piece - which is what you want since they should have tails on them! -- Dr. B
100b lane 1, lanes 8 and 9 are the premade primers and the oligioprimers respectively
Lanes 2 -5 are Max's samples. Both lane 8 and 9 used the oligioprimers and the first and last primers of the deep well box to initiate the pcr reaction. Both were successful in that they had the right size for bp so I will continue on using the oligoprimers as they seem to have a brighter band.
Week2:
9.13.12
3rd secondary PCR using Phusion and new Lmstpp Primer from concentrate
9.11.12
Ran secondary PCR 2nd time, no results
Week1:
Primary (lane 3) and secondary (lane 4)PCR with 1 kb ( lane1) and 100 b (lane 2) ladder
Due to the change from KOD polymerase to Q5 this week was spent restarting overlaps and transitioning to the new polymerase. My signal in the 4th column for the secondary PCR with Plic forward and reverse primer displayed weak amplification. Possible reasons could be the new polymerase, the slightly altered temperatures for the Q5 thermocycler program affects the primers, or that the primers that I used were old/defective. In any case, since the primary shows a good smear, I'll redo the secondary with the same reagents and find a new plic for/rev sample.
Week 7:
After sending dna samples to the lab for sequencing, the results did not match with my target after doing a blast comparison of the sequence. I will restart the experiment from scratch and attempt inserting the target once again.
Week 6:
7.19.12
PCR clean up protocol and concentration by nanospectrometer:
Final concentration of plasmid : 26.7 ng/Ul
7. 18.12
Preformed primary and secondary PCR for leishmania major
Gel of Leishmania major: Lane 1 1kb ladder, lane 2 Primary pcl with smear, lane 3 secondary overlap pcr with distinct bands. The rest of the lanes are another person with the same targer
7.17.12
Worked on primer design and Bsa-4 primer design and found all necessary reagents .
Week 5:
Spent the week work on pymol 1,2, and 3.
Started reading and looking into Primer design and Bsa-4 primer design
week 4:
7.6.12 Pnic-Bsa 4 PCR results 2
Lane 1: 100 bp marker, Lane 2: 1/10 Pnic, Lane 3 1/100 Pnic, Lane 4: 1/1000 Pnic, lane 5: No Dna control
Note:
Better results than the first PCR reaction. Bands are not clear/ there is bad resolution. As well there is evidence of PCR in the no DNA control lane.
Possible reasons for error:
Lanes 2 and 3 could have contaminated lanes 1 and 4. This would be a good reason why the middle 2 lanes have very weak signals while the outer 2 lanes have very strong signals.
7.5.12 Pnic-Bsa 4 PRC results 1
Lane 1: 100 bp marker, Lane 2: 1/10 Pnic, Lane 3 1/100 Pnic, Lane 4: 1/1000 Pnic, lane 5: No Dna control
No polymerization chain reaction detected. Possible reasons:
The wrong concentration of dNTP was added
Wrong primers were added
Inadequate amount of DNA was added
7.3.12 PmCherry PCR results
:
Note: Unfortunately Taq Pol was left out of the first 4 tubes by accident. To describe the No Dna control of lane 9, it is probably that the results are due to contamination from lane 8.
Week #:
070212 - Paul - good reads on the pNIC_bsa4 sequencing - can you figure out what gene is present in the original vector here? -- Dr. B
6.28.12 Results of DNA sequencing :pNIC-Bsa4
Gene found : Bacillus subtilis sacB gene for levansucrase
sac B is a counter selection marker.
Lavansucrase catalyzes the reaction: sucrose + (2,6-beta-D-fructosyl)n
12.6.12
Worked with ling to assay suman's protein.
The measured absorbance for 410nm was 0.
Since other people had similar results it could be reasoned that the cause for this would be:
1.multiple times of defrosting
2.the glycerol mix was bad
3. it was not stored in glycerol at all
Afterwards we measured Suman's protein STSP 2 more times. the first time it did not work because the values for the uM and molecular weight were incorrect, so the addition of tris base was incorrect. The second time, the correct values were entered, and that is when the above conclusions were made.
12-5-12
Enzyme assay with FtHap; Negative results as there is no absorbency measured at 410 nm which is the wavelength where the protein should present itself if there was indeed protein activity when introduced with the pNPP.
reasons could be :
1. improper technique with pipetting and use of the wrong cuvette
2. the protein was inactive due to denaturing
3. not enough of the protein within the solution.
The values (absorbance) were too small to consider using for inhibition. During the collection of absorbance, the samples got mixed up. Also there isn't a linear function as the concentrations of enzyme increased. Also, one of the samples was thrown out because it was skewed from the rest of the data.
12-4-12
Conducted enzyme assays with ling and rishi on the surrogate FtHap. Still getting inconclusive results with the redtide machine. More assays will be run in order to verify whether or not the issue is technique or the protein.
Conducted virtual screening 2nd run - results:
The results shown here are the top 10 ligands selected by the GOLD program out of 400 compounds from the HF9PlatesPlates library. Compounds HTS09290, SEW02685, and HTS08822 are the top 3 ligands from the selection of 400 and would make the best potential drug candidates out of the compounds screened. The compound 1 shows much promise with a fitness score of 99.23%.
Week 14:
11.30.12
Started work on virtual screening.
11.29.12
Worked on assaying / measuring concentration samples
Worked on swiss model:
11.27.12
Worked on purification protocols - used GE purification tubes in combination with the beckman centrifuge in order to remove water and extract a pure sample. Sample found to be roughly around 0.98 mg/ml.
week 13:
112612 - ?? . Dr B
Characterized the surrogate with rishi -
Nanodrop/concentration pulled from rishi's vds page.
Lane 1: Skipped
Lane 2: 10-170kDa Protein Ladder
Lane 3: Sample 1, Cell lysate after induction
Lane 4: Sample 2, Soluble fraction
Lane 5: Sample 3, Flow through
Lane 6: Sample 4, Wash
Lane 7: Elution 1 from Trial 1 (Tube A)
Lane 8: Elution 2 from Trial 1 (Tube B)
Lane 9-10: Skipped
Some of Elution 2 spilled into Lane 9 causing faded bands. Also, Cell Lysate before induction was not inserted into Lane because not available.
Elution 1 (Concentration of 1.40 mg/mL): Tube A from Trial 1
Elution 2 (Concentration of 0.11 mg/mL): Tube B from Trial 1
Elution 1 (Concentration of 1.08 mg/mL): Tube C from Trial 2
Elution 2 (Concentration of 0.10 mg/mL): Tube D from Trial 2
Week 12:
11.18.12
Assisted Rishi on Sonification steps.
Out put from the sequencing of lmstpp target:
"
When entered onto blast, there was not enough of a match up to give conclusive results.
Week11:
Paul - make sure you use the right primers for you sequencing. Not sure why you are getting unreadables. Maybe show me you output form one of them - post it here on Wiki. -- Dr. B 11/19/12
11.15.12
Morning to mid day was spent with the continued growth of the Fthap colonies in 2L flask ; 4 out of the 6 flasked were used due to space constraints. Every hr I measured OD for the flasked until 0.5 absorbency at 600 nm was reached. Afterwards I added ITPG and left the flask in the shaker for 4 hours to allow it to grow. Subsequent centrifugal was done by ling and rishi after the 4 hour period.
11.14.12
I stayed over night with ling to work on growing up the fthap colonies for surrogate protein expression. 6 flasks in total were left in the shaker over night.
11.12.12
I looked over the data recieved from the core and checked against blast was inconclusive. There was a large number of N and unreadable nucleotides in the sequence. I will grow more bacteria this week and resend samples for sequecing to verify my results.
As well I will now start on the surrogate protein expression.
Week10:
11.9.12
I am currently awaiting results of DNA sequencing.
11.8.12
Nanodrop concentration results : Samples 4 and 9 show really weak absorption/concentration. The reasons for this could be that those colonies did not grow too well in the media, improper growing technique, or improper minipreping.
Next step would be to prepare the samples for sequencing.
11.7.12
Worked on miniprep of the colonies and extraction of dna.
11.6.12
I prepared 9 colonies for master plate production and grew up 9 tubes.
My everything plate has produced colonies - there are roughly around 60 colonies. My next step is to move onto picking a couple of colonies and proceed to making a master plate and producing some bacteria for sequencing.
Week9:
11.2.12
I have decided to employ a hail mary approach with my own samples while working with ling and rishi to help them get their samples to cloning. What I have done is mixed together 3-4 months worth of my own personal leftover and unused lmstpp and bsa-4 samples that were properly labeled into one tube of accepting plasmid and one tube of the insert. This produced a high concentration of plasmid to insert. I also ran a gel check to see if the samples were still use able. On a 100b ladder, both the insert and the accepting vector seem to be within the right size, length, and cut amounts. When plating I made a 20:20 ratio of accepting to insert - and added 50 ul of the bacteria to the mixture while prepping. As well checking the nanodrop readings, while the absorbances are high, the wavelengths they each respectively peak at are correct when compared to past samples.
11.1.12
Worked on PCR^2 and clean up with ling.
10.31.12
Made primer dilutions for the master stock and for working solutions. Attempted to do a midiprep clean up - failed horribly due to incorrect tubes being used.
10.29.12
Prepared samples for additional people for leishmania target.
week8:
10.25.12
Checked on the plates created - no colony growth present. I will attempt to start back at the plasmid and insert preparation step.
10.23.12
By the end of this day I was able to clean up the pcr samples, prepare the plasmid, and transform and plate the bacteria.
10.20.12
Week 7:
102112- Paul, ok - show some data/images. -- Dr. B
10.18.12
Worked on PCR^2 ; I created 16 samples, half for gel extraction and the other half for regular pcr clean up procedure.
10.16.12
continued work on the virtual refresher assignment.
10.15.12
Worked on acquainted self with virtual screening program. Looked over and discussed results of the DNA sequencing and compared it with the online nucleotide BLAST program. Out of the 14 samples sent only 3 had any sort of match up with the gene of interest. Of the three none of the samples had the right size to continue onto the next phase - it can be assumed that only a fragment of the insert actually got into the plasmid. Due to this I will have to restart back at the secondary PCR procedure and try to adjust the protocol to see if theres any change in results.
Week 6:
101612 -Paul - ok good job. Hopefully your cloning works out! - Dr. B
10.13.12
Currently working on virtual screening protocols.
10.12.12
The cleaned up plasmids had concentrations determined through the use of the nanodrop machine. The ranges are from 10-22 ng/ul for plasmid concentration. Concentration seems medium to low . 14 samples were sent to sequencing. 12 were had using the forward primers, and the 2 samples with the highest concentration were used with the reverse primers. Still awaiting results.
10.9.12
Colonies were grown up in snap tubes and grown up over night. Procedures for the day included spinning down and using the miniprep clean up kit to extract the plasmid.
10.8.12
Plates grew up successfully over the weekend , a nice amount of colonies are present on each of my plates with out too much of a lawn. Colony count seems to be in the 10's. I will proceed to master plating and growing up in tubes.
Week 5:
100912 - Paul - I think that your PCR^2 bands need to be a lot bigger in order to have enough to go on to cloning. Does the gel of PCR^2 correlate to the amounts that you get in Nanodrop. Because the nanodrop vaules seem good for the PCR^2 but the bands are weak. Also, you should have multiple wells of PCR^2 - not just one or two , right? - DR. B
Also, try to crop your gels better to get rid of the black space.
10.3.12 ( BSA4 cutting and prepartation - I made 2 preparations of the solution so I could make 2x the plates)
10.2.12 ( I redid my PCR^2 results on gel)
Week 4:100112 - Paul, yeah - make sure there is something there before proceeding. -- Dr. B
9.25.12
Week3:
paul - Great. And the 'tail primers' on the left seem to make a larger piece - which is what you want since they should have tails on them! -- Dr. B
Lanes 2 -5 are Max's samples. Both lane 8 and 9 used the oligioprimers and the first and last primers of the deep well box to initiate the pcr reaction. Both were successful in that they had the right size for bp so I will continue on using the oligoprimers as they seem to have a brighter band.
Week2:
9.13.12
9.11.12
Week1:
Due to the change from KOD polymerase to Q5 this week was spent restarting overlaps and transitioning to the new polymerase. My signal in the 4th column for the secondary PCR with Plic forward and reverse primer displayed weak amplification. Possible reasons could be the new polymerase, the slightly altered temperatures for the Q5 thermocycler program affects the primers, or that the primers that I used were old/defective. In any case, since the primary shows a good smear, I'll redo the secondary with the same reagents and find a new plic for/rev sample.
Week 7:
After sending dna samples to the lab for sequencing, the results did not match with my target after doing a blast comparison of the sequence. I will restart the experiment from scratch and attempt inserting the target once again.
Week 6:
7.19.12
PCR clean up protocol and concentration by nanospectrometer:
Final concentration of plasmid : 26.7 ng/Ul
7. 18.12
Preformed primary and secondary PCR for leishmania major
7.17.12
Worked on primer design and Bsa-4 primer design and found all necessary reagents .
Week 5:
Spent the week work on pymol 1,2, and 3.
Started reading and looking into Primer design and Bsa-4 primer design
week 4:
7.6.12 Pnic-Bsa 4 PCR results 2
Note:
Better results than the first PCR reaction. Bands are not clear/ there is bad resolution. As well there is evidence of PCR in the no DNA control lane.
Possible reasons for error:
Lanes 2 and 3 could have contaminated lanes 1 and 4. This would be a good reason why the middle 2 lanes have very weak signals while the outer 2 lanes have very strong signals.
7.5.12 Pnic-Bsa 4 PRC results 1
No polymerization chain reaction detected. Possible reasons:
The wrong concentration of dNTP was added
Wrong primers were added
Inadequate amount of DNA was added
7.3.12 PmCherry PCR results
:
Note: Unfortunately Taq Pol was left out of the first 4 tubes by accident. To describe the No Dna control of lane 9, it is probably that the results are due to contamination from lane 8.
Week #:
070212 - Paul - good reads on the pNIC_bsa4 sequencing - can you figure out what gene is present in the original vector here? -- Dr. B
6.28.12 Results of DNA sequencing :pNIC-Bsa4
Gene found :
Bacillus subtilis sacB gene for levansucrase
sac B is a counter selection marker.
Lavansucrase catalyzes the reaction:
sucrose + (2,6-beta-D-fructosyl)n
Forward:
NNNNNNNNNNNNNNNGGNNNNNNANNNNTGTACTTCCATCNATGGAGACCGACGTCCACATATACCTGCCGTTCACTANN NNNNGTGAAATGAGATATTATGATATTTTCTGAATTGTGATNNNNNNGGCAACTTTATGCCCATGCAACAGAAACTATAA AAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGNNNNNNNGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCT ATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGG GTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTT TAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTAC ATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCT GGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTA CACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATAAAGTTCCTGAGTTCGATTCGTCCACAATT AAAAATATCTCTTCTGCAAAAGGCCTGGACNTTTGNGACAGCTGGCCATTACAAAACACTGACNGCACTGTCGCAAACTA TCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAA AAGTCGGCGAAACNTNCTATTGACAGCTGGAAAACGCTNGGCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGA TTCTATCCTAAAGACCAAACNCAANNATNGGTCNGGTTCANCCNCNTTTACATCTGACGGAAAATCCGTTTATTCTACAC TGATTNNTCNGNAAANATTNNGGNAAACNAANNNCTGANANTGCNCAAGNNNNNTATCANCNNCANAANNGCNNTNNNAN NNANGGNGNNNNANGNNNNNNANNANCNTTGNNNGNNNNNGNNAANNTNNNCNAANNNNNNNCNNNNNTCNNNNNANNNC NANNNNCNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNTNNNNNNAANNANNNNNNAGNNNNNNCNNNNNNNNNN
Reverse:
NNNNNNNNNNNNNNNNGNNNNNNGTGNNCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATCCGTATCCAC CTTTACTGGAGACCGTCAATGCCAATAGGATATCGGCATTTTCTTTTGCGTTTTTATTTGTTAACTGTTAATTGTCCTTG TTCAAGGATGCTGTCTTTGACAACAGATGTTTTCTTGCCTTTGATGTTCAGCAGGAAGCTAGGCGCAAACGTTGATTGTT TGTCTGCGTAGAATCCTCTGTTTGTCATATAGCTTGTAATCACGACATTGTTTCCTTTCGCTTGAGGTACAGCGAAGTGT GAGTAAGTAAAGGTTACATCGTTAGGATCAAGATCCATTTTTAACACAAGGCCAGTTTTGTTCAGCGGCTTGTATGGGCC AGTTAAAGAATTAGAAACATAACCAAGCATGTAAATATCGTTAGACGTAATGCCGTCAATCGTCATTTTTGATCCGCGGG AGTCAGTGAACAGGTACCATTTGCCGTTCATTTTAAAGACGTTCGCGCGTTCAATTTCATCTGTTACTGTGTTAGATGCA ATCAGCGGTTTCATCACTTTTTTCAGTGTGTAATCATCGTTTAGCTCAATCATACCGAGAGCGCCGTTTGCTAACTCAGC CGTGCGTTTTTTATCGCTTTGCAGAAGTTTTTGACTTTCTTGACGGAAGAATGATGTGCTTTTGCCATAGTATGCTTTGT TAAATAAAGATTCTTCGCCTTGGTAGCCATCTTCAGTTCCAGTGTTTGCTTCAAATACTAAGTATTTGTGGCCTTTATCT TCTACGTAGTGAGGATCTCTCAGCGTATGGTTGTCGCCTGAGCTGTAGTTGCCTTCATCGATGAACTGCTGTACATTTTG ATACGTTTTTCCGTCACCGTCAAAGATTGATTTATAATCCTCTACACCGTTGATGTTCAAAGAGCTGTCTGATGCTGATA CGTTAACTTGTGCAGTTGTCAGTGTTTGNTTGCCGTAATGTTTACCNGANAAATCANTGTAGAATAAANGGATTTTTCNT CNGANGTNAATGNGNTGNNNGACNTNNNNNNTNNNNNTTTNNNNNNANCATTGCATCNATTGTCNCNNNCTTNNGANNNN NNNNNTTTTNCNNCNNNNANNNANNTNNNNNANTTNNNNNNANNNNNNNNNANNNNNNNCANCNNNTNNNNNNNNNNNNN GNNNNNNNNNNNNNNNNGNNNNNNNNNNNNNNNNN
======================================================
6.28.12 Gel of PCR of PBGR22
===================================================================
6.26.12 DNA sequencing results of PMCherry:
Forward:
NNNNNNNNCNNNNCGAGGANGNNAACATGGCCNTCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTG AACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGAC CAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACC CCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGC GGCGTGGTGACCGTGACCCAGGACTCCTCCTTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTT CCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCG CCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAG GCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACAC CATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAATAATACTAGA GCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCT CTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAA AAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCA CTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTANTACGGTNNTCCACAGAA TCNNGGNANANNCNNNANNANNTGTGAGCAAAAGNCAGCAAAAGNNGNNNTAAAANNNCNTTGCTGNNTTTTCNNNGNTC NCCCCNGANNNNNNTNNNNAAANCGANNNTNANNNNAANGNGNNNNNNNNNNNNNTNNNNNNNNCNGNNTTNCCCNNNNN CNNNNNNNNNNNCNNNNNACNNNNNNNCNNNNNNNNNNNNNTTNNNCNTNGNNANNNNNNNNNNNN
The pmCherry vector tested is indeed mCherry as indicated by a BLAST search.
Cloning vector pME-mCherry-T2A-EGFP, complete sequence Length=4107
Score = 1232 bits (667), Expect = 0.0 Identities = 678/684 (99%), Gaps = 0/684 (0%) Strand=Plus/Plus Query 1 AACATGGCCNTCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAAC 60 ||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 698 AACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAAC 757 Query 61 GGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACC 120 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 758 GGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACC 817 Query 121 GCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCT 180 ||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||| Sbjct 818 GCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCT 877 Query 181 CAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTG 240 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 878 CAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTG 937 Query 241 AAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGC 300 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 938 AAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGC 997 Query 301 GTGGTGACCGTGACCCAGGACTCCTCCTTGCAGGACGGCGAGTTCATCTACAAGGTGAAG 360 ||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||| Sbjct 998 GTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAG 1057 Query 361 CTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGG 420 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1058 CTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGG 1117 Query 421 GAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAG 480 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1118 GAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAG 1177 Query 481 AGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCC 540 ||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||| Sbjct 1178 AGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTCAAGACCACCTACAAGGCC 1237 Query 541 AAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCC 600 ||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||| Sbjct 1238 AAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGCTGGACATCACCTCC 1297 Query 601 CACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACC 660 ||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||| Sbjct 1298 CACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCGCCGAGGGCCGCCACTCCACC 1357 Query 661 GGCGGCATGGACGAGCTGTACAAG 684 |||||||||||||||||||||||| Sbjct 1358 GGCGGCATGGACGAGCTGTACAAG 1381
Week 2:
PGFP DH5a E. Coli on Amp. colony plates (6.22.12)-
Colony A - 1 ng/ul ; 1 colony ; 1 efficiency
Colony B - 5 ng/ul ; 544 colonies ; 108.8 efficiency
Colony C - 25 ng/ul of plasmid; 29 colonies ; 1.16 efficiency
==============================================
===================================================
DNA sequencing results ( 6.21.12) pGBR22Forward:
NNNNNNNNNNNNNNNGNNNNNNNCCGACGTCGCATGCTCCGGCCGCCATGGCCGCGGGATTTTAGTGATGGTGATGGTGA
TGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATC
CAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACAAAT
AGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGTG
TTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTA
GATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCCC
ATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTAT
TGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCCC
CTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGTANG
TCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCGACC
ATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTNAAATAGCTTGGCGTAATCATG
GTCATAGCTGTTTCCNGTGTGAAATTNTTATCCGCTCACAATTCCNCACNACATACNAGCCNGAAGCATAAANNGTAAAG
CCTGGGGTGCCTAATGAGTGANNTAACTNNNATTAANTGCNNNGCNCTCACTNCCCGNTTTCCAGTCGGGAAACCTGTCN
NGCCNNCTGCATTAATGAATCNGGCNANNNGCGGGNNANNNNNTTNNCGTATGGNNNCNNTTCCGNTNNNNNNNNNNNGN
ANTCGNTNNNNNGNNNNTNNNNNNNNNNNNCGGTATNNNNTNNNTNNAGGNGNNNNCNGNNNNNCNNNNNNNNNNNNNNN
CCNNNNNANNNNNNNNNNNNNNNCNNNCNANNGNNNNNTNNNNCNNNNNANNNNNNNNNNNNNNNNNNNNNNN
Reverse:
NNNNNNNNNNNNNNANNNTAGNNTACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGC
GGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATA
TGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTA
AAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACC
ATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGA
ACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATC
TCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTT
TGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCA
AATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCAC
AACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCA
CTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCA
CTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGNGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTT
CGCCAGCTGGCGTAATAGCGAANAGGCCCGCNNCGATCGCCCTTCCNANAGTTGCGCAGCCTGANGGCGAATGGNCNCNN
CCNGTAGCGNGCATTNAGCNNNGNGGGNNNNGGNGGNTACNCNCAGCGNNACNCTANNNNNNNGCNCCNNNNNCNNNNNN
NTTTNNNTTNNNNNNNNNNNNNCNNNNNNCGNTTCCNNTNANNNNNNNNNGGGGNTNNNNNNNNNATTNNNNNNTNNGNN
NNNNNNNNANNTGATNGGNNNNNNNNNNNNNNNNNGNNNNNNNNNNNNNN
====================================================
Dna extraction of pmCherry Plasmid, nanodrop sample (6.21.12) : 209.9 ng/ul
Week 1: